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Probiotics and Inflammatory Bowel Disease

Gordon S. Howarth1,2,3,4,5,6

1Centre for Paediatric and Adolescent Gastroenterology, Children, Youth and Women’s Health

Service; Disciplines of 2Physiology, 3Paediatrics, and 4Agricultural and Animal Science, University

of Adelaide; 5School of Pharmacy and Biomedical Sciences, University of South Australia;

6School of Biological Sciences, Flinders University, Adelaide, South Australia.

Short Title: Probiotics and Inflammatory Bowel Disease.

Correspondence: Associate Professor Gordon S. Howarth

Centre for Paediatric and Adolescent Gastroenterology

Children, Youth and Women’s Health Service

72 King William Road

North Adelaide, South Australia, 5006, Australia

Email: gordon.howarth@adelaide.edu.au

Tel: 61-8-8161-6872

Fax: 61-8-8161-6088

mailto:gordon.howarth@adelaide.edu.au

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Abstract

The aetiology of inflammatory bowel disease (IBD) remains obscure, currently thought to be

associated with a genetic predisposition, dysregulation of the mucosal immune system, and a

loss of antigen tolerance to enteric microflora, further influenced by a range of other

environmental factors. In many cases, disease activity can be unremitting and refractory to

treatment, with an unpredictable response to conventional therapy. To this end, new treatment

strategies are being pursued on the basis of our understanding of IBD pathogenesis, and there

is increasing evidence that at least some components of the enteric flora are primary

contributors. Restoring the balance of the colonic microbiota to a less-pathogenic state is

therefore a desirable strategy. Probiotics are currently defined as live non-pathogenic micro-

organisms that, when ingested, exert a positive influence on host health beyond basic

nutrition. On this basis, probiotics hold the potential to restore normal intestinal homeostasis.

Despite more than a century of anecdotal reports of probiotic efficacy in gastrointestinal

disease, only relatively recently have well-controlled, scientific studies and clinical trials,

been conducted. Whilst reliable in vitro predictors of potential in vivo efficacy of putative

probiotics await development, well-characterised animal model systems are proving valuable

for the methodical, pre-clinical development of probiotics. Although early probiotic

applications focussed largely on lactic acid bacteria (lactobacilli) and bifidobacteria, the

range of candidate probiotics has now expanded significantly. Successful clinical application

of the probiotic formulation, VSL#3, for treatment of the pouchitis variant of IBD, has

instilled new excitement into the applicability of probiotics to IBD treatment, and the

potential importance of probiotic combinations. The availability of new recombinant

methodologies to develop ‘designer’ probiotics, capable of synthesizing and secreting specific

factors, ranging from vitamins through to antibodies, further broadens the scope for probiotic

application in IBD. Indeed, there are encouraging reports that probiotics may not need to be

viable, or even intact, to exert their beneficial effects, with reports of therapeutic benefit from

bacterial components such as DNA. In addition to the development of rigorous predictive

systems to ascertain probiotic efficacy, challenges for the future will include determining the

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optimal probiotic, or probiotic combination, and its timing of administration during phases of

IBD relapse and remission. At present, our understanding of the intestinal microflora, and the

importance of its composition and variability between individuals, is limited. However, once

this understanding has been attained, strategically-designed probiotic formulations could

ultimately be ‘tailored’ to suit individual IBD patients.

Introduction

Inflammatory bowel disease (IBD) is the collective term for a group of chronic, idiopathic

inflammatory disorders that affect the gastrointestinal system. Crohn's disease and ulcerative

colitis (UC) are the most common and serious variants of IBD, together imposing significant

patient morbidity, economic burden, and long-term healthcare dependence. The aetiology of

inflammatory bowel disease (IBD) remains obscure, currently thought to be associated with a

genetic predisposition, dysregulation of the mucosal immune system, and a range of other

environmental factors (1). In many cases, disease activity can be unremitting, with an

unpredictable response to conventional therapy.

Considerable progress has been made in studies of IBD genetics over the last decade, and the

complementary strategies of genome-wide scanning and candidate gene-directed studies have

led to the identification of a number of genetic markers that appear to predict disease

susceptibility and behaviour (2). Identification of genetic markers that predispose to

inflammation (alleles DR2, DRB1*0103 and DRB1*12) and a Crohn’s disease susceptibility

gene on chromosome 16 [NOD2/CARD 15: Nucleotide-binding oligomerization domain

protein 2] has paved the way for exciting new developments in therapeutic approaches for

IBD (3,4). NOD2/CARD 15 is a cytosolic protein involved in intracellular recognition of

microbes by sensing peptidoglycan fragments, and there is compelling evidence that it serves

as an intracellular pattern recognition receptor to enhance host defence by inducing the

production of antimicrobial peptides such as human beta-defensin-2 (5).

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The characterisation of newly-identified NOD2/CARD-15 mutations is providing new

information on the likely contribution of a defective host anti-microbial defence system to

IBD aetiology and pathogenesis. This could have exciting implications for the use of

candidate micro-organisms as a novel treatment strategy for IBD. Prospectively, this

information would be particularly valuable when combined with a concomitant strategy to

address the genetic basis of responsiveness to IBD therapy. Indeed, the spectrum of new

treatment modalities for IBD has expanded exponentially in recent years (reviewed in 6).

These genetic and genomic strategies could form the basis for better predicting the likelihood

of responsiveness to newly-developed IBD therapies associated with the utilisation of micro-

organisms, or factors derived from such organisms.

Probiotics

Although under continual review, the generally agreed definition of probiotics encompasses

"live micro-organisms which, when consumed in adequate amounts, confer a health benefit

on the host beyond basic nutrition" (7). This definition, however, may prove to be too limited

and the term ‘alimentary pharmabiotics’ has been coined to further encompass dead

organisms, or bacterial constituents which may be genetically or otherwise modified, and may

not necessarily be restricted to those of human origin. Lactobacilli (8,9) and bifidobacteria,

(10) species are generally referred to as archetypical probiotics, with increasing reports of

probiotic properties attributed to non-pathogenic Escherichia coli (11,12) and non-bacterial

organisms, such as Saccharomyces boulardii (13). Probiotics are thought to function through

a number of different actions including: the production of antimicrobial agents such as

bacteriocins, hydrogen peroxide and organic acids; blocking adhesion of pathogens or toxins

to epithelial cells; providing antioxidant agents; and modulation of the immune system

(14,15). Although the current review focuses on probiotic effects in the context of IBD, the

complex interplay of mechanisms associated with specific organisms, or combinations of

probiotic organisms, extends to applications for a broader range of medical disorders.

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Probiotics and IBD

Perhaps not surprisingly, the idiopathic nature of IBD, combined with its profound

inflammatory characteristics, has resulted in current therapeutic strategies being targeted,

almost exclusively, at disease immunomodulation (16). Notwithstanding this historical

approach, new treatment modalities are now being pursued on the basis of our increased

understanding of IBD pathogenesis. To this end, there is accumulating evidence that at least

some components of the enteric flora are primary contributors to the unrelenting intestinal

inflammation so characteristic of IBD. In general terms, restoring the balance of the colonic

microbiota to a ‘less-pathogenic’ state would appear a desirable preventative, or therapeutic,

strategy. However, before we consider probiotics as a potential therapeutic option in IBD, we

should consider the resident enteric microflora and the potential impact of probiotic

organisms in the context of IBD aetiology.

The intestinal microbiota and IBD aetiology

Although the intestinal flora is essential for host defence, some of its constituents may, in

genetically susceptible hosts, become a risk factor for IBD, and it has been proposed that

resident bacterial flora play a pivotal role in IBD pathogenesis (17). Mechanisms underlying

the influence of the intestinal flora on mucosal homeostasis, mucosal protection, development

and function of immune responses, and metabolism of fecal residue are undergoing increased

scientific scrutiny. Strategies to enhance the beneficial properties of endogenous microflora,

or alternatively, to minimise deleterious effects, represent a logical therapeutic approach,

forming the basis for manipulation of the intestinal flora in IBD treatment (18).

Nevertheless, it is important to understand that an inappropriate reaction to infection by a

specific persistent pathogen has not yet been eliminated as a possible aetiological component

in IBD. Indeed, Crohn’s disease and UC share histopathological similarities with defined

intestinal infections, and occur in areas with highest luminal bacterial concentrations (17,18).

Strengthened by the causative link between Helicobacter pylori and gastric ulceration (19),

many microbial pathogens have been promoted as initiators and perpetuators of IBD. Perhaps

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Mycobacterium avium, subspecies paratuberculosis (MAP) provides the most persuasive

evidence as a causative agent in IBD since it manifests a Crohn’s-like phenotype in livestock

in the condition known as Jonne’s disease (20). Other potential pathogenic causes have

included M. kansaii, Escherichia coli, Diplostreptococcus species, viral vectors (measles,

RNA viruses), Listeria monocytogenes, Fusobacterium necrophorum, Chlamydia species,

Pseudomona maltophila and Helicobacter hepaticus (21,22,23). Taken together, identified

probiotics capable of altering the intestinal environment such that colonization by the

aforementioned species is inhibited, would therefore appear to be a logical strategy in IBD.

Antibiotics have a defined role in the management of IBD and its complications, although

their long-term usage is undesirable due to the risk of toxicity, bacterial resistance and

overgrowth. Nevertheless, it would be fair to state that the current weight of scientific

literature identifying a single organism as causative of IBD, although highly desirable, is not

particularly compelling. Although several authorities, including the Joint Food and

Agricultural Organization of the United Nations and the World Health Organization (24,25),

have described selection criteria for probiotic organisms, to date, no reliable in vitro

predictors of in vivo efficacy of putative probiotics have been identified. This has been

compounded by the realization that individual probiotics do not appear to act through a single

mechanism, further complicated by the likelihood that probiotic combinations, as opposed to

specific candidates, may be indicated for certain disorders. Indeed, the mechanism of action

of probiotics is likely to vary with different strains and may also be dependent on the clinical

condition for which it is applied. Although well-characterised animal models are gaining

momentum as predictive pre-clinical efficacy systems, a discussion of likely probiotic

mechanisms is indicated.

Mechanisms of probiotic action in IBD

Although perhaps a little simplistic, the ability for probiotics to prevent or combat infections

may be partially- or entirely dependent upon mutual competitive metabolic interactions with

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potential pathogens, production of anti-microbial peptides such as bacteriocins (25) or

inhibition of epithelial adherence and translocation by pathogens (26,27). Interestingly,

Collado et al (28) reported that in general, bifidobacterial strains of animal origin

demonstrated an improved capacity to inhibit and displace pathogens from human mucus than

were human strains, further reporting that bifidobacteria were capable of producing

antimicrobial peptides directed against Helicobacter pylori (29). Cross-species utilization of

probiotics may therefore provide another important level of complexity for probiotic utility in

clinical disorders such as IBD. In certain allergic disorders, including atopic eczema,

probiotic influences on mucosal barrier function may be operative (30), whilst multiple

mechanisms may account for anti-neoplastic effects (31).

Probiotics, tolerance and the immune response

Since control of bowel inflammation has generally been recognised as paramount in IBD, it is

perhaps not surprising that down-regulation of mucosal inflammation has been identified as

the primary focus of both conventional and probiotic-targeted therapy, fuelled by the

cumulative clinical experience with anti-TNF based treatment successes (32).

Rodents and humans are normally tolerant to autologous microbiota, and an association

between breakdown of this tolerance and the development of chronic intestinal inflammation

has been demonstrated (32). Potentially, pathological responses to components of the

intestinal flora may occur under normal physiological conditions, but these may be

suppressed by immunoregulatory mechanisms. In a recent review by Thompson-Chagoyán et

al (33), 11 models of IBD have been described, in which inflammation was found to be

dependent on the presence of normal flora; the absence of normal flora being associated with

non-appearance of the condition (34,35,36,37). This phenomenon has been reported across

species (mice, rats and guinea pigs), and to occur in manipulated organisms such as

transgenic mice with targeted deletion of the T-cell receptor (TCRα), that spontaneously

develop colitis in response to the gut microbiota (38). Mucosal inflammation in rats and mice

with induced IBD has also been reported to respond to treatment with broad-spectrum

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antibiotics (39). Moreover; in some animal model systems, colonisation with normal flora

results in the rapid development of T-cell-mediated gut inflammation which can be

transferred to other animals using activated T cells directed against enteric bacteria (40).

Nevertheless, it appears that not all commensal bacteria have an equivalent ability to induce

mucosal inflammation, which is also influenced by the host genetic background.

Following cell surface recognition via Toll-like receptors (41), the anti-inflammatory effects

of probiotics require signalling with the epithelium and hence, the mucosal immune system

(42). Although transduction of bacterial signals into host immune responses presumably

involves more than one pathway, NF-kappaB has been established as a central regulator of

epithelial responses to invasive pathogens (43,44). Non-pathogenic components of the flora

may attenuate pro-inflammatory responses by delaying degradation of IkappaB, the counter-

regulatory factor to NF-kappaB (44). Indeed, it may be that probiotic bifidobacteria and

lactobacilli may not use the same mechanism to achieve their anti-inflammatory effects as

other signal transduction pathways begin to emerge.

Clearly, a better understanding of the interplay between prokaryotes and eukaryotes, and

subsequent effects on metabolic activity, will facilitate our knowledge of probiotic

mechanisms. Bacterial adjuvants, including peptidoglycan, lipopolysaccharide, and DNA

(CpG) bind to membrane-bound Toll-like receptors (TLR-2, 4, and 9. respectively), or

cytoplasmic (NOD1 and NOD2) receptors (pattern recognition receptors), that activate

nuclear factor-kappaB and transcription of many proinflammatory cytokines (45). Prokaryotic

DNA perhaps represents the first description of a growing number of bacteria-sourced factors

with the potential to alter host epithelial and mucosal immune responses. Un-methylated

cytosine–guanine (CpG)-containing DNA, the ligand for Toll-like receptor 9 (TLR9), is a

recently recognized microbial product with immuno-stimulatory and immuno-regulatory

effects (46). TLR9 is expressed by many cell types located in the intestine, including

epithelial cells and dendritic cells, and subcutaneous administration of immuno-stimulatory

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DNA has been reported to reduce the severity of experimental and spontaneous colitis in

murine models of IBD (47) via a mechanism attributed to TLR9 signalling (48).

A decrease in the secretion of pro-inflammatory cytokines, IFN-gamma, TNF-alpha and IL-

12, and interference with bacterial adherence to the epithelium has been demonstrated

following probiotic administration, associated with NF-kappaB inhibition, heat-shock protein

induction and proteasome inhibition, although NF-kappaB induction has also been

demonstrated (49,50). Unexpectedly, many of these beneficial effects have been achieved not

only by live bacteria, but also by gamma-irradiated non-viable bacteria, bacterial DNA

components and probiotic-cultured media (49,51). Investigations into probiotic supernatants

and their therapeutic potential in IBD are therefore forming the basis for new directions in

IBD research. In summary, although mechanisms of probiotic action may vary, depending on

the experimental or clinical context, and depending on differences in the host and in the

bacterial strain, the engagement with host immunity is pivotal to probiotic action in IBD.

Probiotics as therapeutic agents in IBD

The human colon is a densely populated microbial ecosystem with several hundred bacterial

species usually present with a total weight estimated to be several hundred grams (52). There

are up to 1013–1014 total bacteria in the human intestinal tract, representing 10- to 20-fold

more than the total number of tissue cells in the entire body, with most bacteria being obligate

anaerobes, including clostridia, eubacteria, bacteroides groups and the genus bifidobacterium,

such as Bifidobacterium bifidum and Bifidobacterium infantis (53).

Probiotic efficacy has been confirmed in animal model studies by several investigators with

different probiotic strains (37,53), although the use of probiotic therapy for IBD has only

recently attracted serious interest from clinicians. Clinical trials in Crohn's disease with

organisms, including lactobacillus (54) yeast (13), and coliforms (56), have been confounded

by small patient numbers, differences in disease activity and variations in disease distribution

(56). These deficiencies have been exacerbated by a growing, but poorly regulated,

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commercial market for probiotics, often linked with somewhat tenuous, or exaggerated,

claims for health benefits. Historically, the stability, optimal dose-range, frequency of

administration and vehicle for delivery, have rarely been determined. However, at present in

Europe, well-designed and appropriately controlled trials of individual probiotic preparations

are underway in both Crohn's disease and ulcerative colitis, to ascertain their efficacy in both

active disease and in prevention of relapse.

Individual probiotics and IBD treatment

The predominant, potentially health-enhancing, bacteria in IBD are the bifidobacteria and

lactobacilli, both of which belong to the lactic acid bacteria (LAB) group (57). These two

genera do not include any significant pathogenic species and their dominance in the faeces of

breast-fed babies is thought to impart protection against infection (58,59). Most commercially

available probiotics meet minimum selection criteria including acid and bile resistance and

survival during gastrointestinal transit, but an ideal individual probiotic strain for any given

indication has still yet to be defined.

Lactobacilli and bifidobacteria have traditionally been the most common candidates for

probiotic-based treatments in IBD. Although there is a growing body of scientific literature

and a wealth of anecdotal information supporting the utility of lactobacillus strains as

therapeutic agents in a range of alimentary disorders, progress has perhaps been hampered by

poorly-controlled associations between probiotic administration and clearly-defined clinical

end-points. Recently, Hawrelak et al (60) conducted a systematic review of six clinical trials

investigating the capacity for Lactobacillus rhamnosus GG to prevent the onset of antibiotic-

associated diarrhoea. As data sources, these investigators employed computer-based searches

of MEDLINE, CINAHL, AMED, the Cochrane Controlled Trials Register and the Cochrane

Database of Systematic Reviews and the bibliographies of relevant papers and previous meta-

analyses. Four of the six trials found a significant reduction in the risk of antibiotic-associated

diarrhoea with co-administration of Lactobacillus GG; one trial reported a reduced number of

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days with antibiotic-induced diarrhoea, whilst the final trial found no benefit of Lactobacillus

GG supplementation. These results are not unique in the context of retrospective probiotic

studies, highlighting the need for additional research and the development of strictly-

controlled predictive systems to clarify the effectiveness of Lactobacillus-based treatments

beyond the effects of LGG in prevention of antibiotic-associated diarrhoea.

Bifidobacterium, a member of the dominant microbiota (i.e. >108–109 colony forming unit

(CFU)/g, represent up to 25% of the cultivable faecal bacteria in adults and 80% in infants.

As probiotic agents, bifidobacteria have been studied for their efficacy in the prevention and

treatment of a broad spectrum of animal and/or human gastrointestinal disorders, such as

colonic transit disorders, intestinal infections, and colonic adenomas and cancer (10). Indeed,

certain strains (eg Bifidobacterium animalis strain DN-173 010) have been reported to

prevent or alleviate infectious diarrhoea through effects on the immune system and resistance

to colonization by pathogens and there is some evidence that certain bifidobacteria may

actually protect the host from pro-carcinogenic activity of intestinal flora (10).

Non-pathogenic Escherichia coli, especially the Nissle 1917 strain, have generated increasing

reports of efficacy in the context of inflammatory disorders. In ulcerative colitis, the non-

pathogenic strain of E. coli Nissle demonstrates efficacy equivalent to that of mesalazine in

ulcerative colitis (61,62). Kamada et al (63) utilising dextran sulphate sodium (DSS)-induced

and IL-10 knock-out models of colitis have recently reported the non-pathogenic E. coli strain

Nissle1917 to prevent both acute and chronic colitis, with its anti-inflammatory properties

attributed not only to viable bacteria but also to heat-killed bacteria or its genomic DNA.

Obermeier et al (64) demonstrated a pro-inflammatory effect of cytosin-guanosin dinucleotide

(CpG)-oligodeoxynucleotide (ODN) treatment in established and chronic DSS-induced

colitis, suggesting that DNA derived from luminal bacteria plays a role in the perpetuation of

chronic intestinal inflammation. These investigators further speculated that treatment with

adenoviral ODN (AV-ODN) could block the known CpG effects in IBD. The apparent

discrepancy between bacterial DNA sources and effects on intestinal inflammation highlights

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the need for well-controlled comparative studies of bacterial DNA, and, more specifically,

CpG motifs, sourced from endogenous or probiotic bacteria.

It should be noted, however, that probiotic properties have not been attributed solely to

bacterial sources, since non-bacterial organisms, such as Saccharomyces boulardii, or even

nematode parasites have been utilised for probiotic purposes (13,65) In summary, it seems

unlikely that a single probiotic will be equally suited to all indications; selection of strains for

disease-specific indications will be required, and it is in this capacity that well-conducted

animal model studies will prove a valuable tool

‘Designer’ probiotics

Genetic modification of food-grade commensal bacteria will need to be accommodated into

our concept of probiotics. To this end, the term "pharmabiotic" has been developed as a more

appropriate generic or umbrella term to encompass any form of therapeutic exploitation of the

commensal flora (66,67). Food-grade bacteria can be modified, or engineered, to achieve

specific functional activity. This can include delivery of anti-inflammatory cytokines or other

biologically active molecules and vaccines to the gut. Of relevance to IBD, the food grade

organism Lactococcus lactis, has been engineered to secrete IL-10 locally within the gut (68).

When tested in two animal models of IBD, the magnitude of this effect was equivalent to

corticosteroid therapy in its ability to decrease inflammation and disease severity. Another

example of the potential applications of engineered commensal organisms is the genetic

modification of lactobacilli resident within the female genital tract to express functional two-

domain CD4 in order to confer protection against HIV infectivity in vitro (69). The future

scope for this strategy is limitless, but public health and other safety concerns must be

resolved before routine clinical use in humans can be instigated. Other examples of

genetically modified (GM) microbes include the delivery of single-chain antibodies for

pathogen-specific passive immunity (70,71) and bacterial-derived trefoil factors to promote

healing and repair in the inflamed mouse gut (72).

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Recently, Inglis et al (73) in a rodent model of intestinal mucositis, utilising the recently-

developed non-invasive sucrose breath test, described a decrease in small intestinal

inflammation following administration of the folic-acid secreting probiotic, Streptococcus

thermophilus TH-4. Although not tested directly, these investigators speculated that the

‘trickle’-delivery of S. thermophilus TH-4 sourced folate to the intestinal enterocytes may

have reduced the severity of intestinal damage induced by the folate depletion, and resulting

inhibition of DNA synthesis, manifest by methotrexate-based chemotherapy. Similarly, Geier

et al (74) have described a partial reduction of colonic inflammation in a rodent model system

utilising Lactobacillus fermentum BR11 reportedly via its unique capacity to modify the

cystine/cysteine equilibrium and hence, reduce oxidative stress.

Public health concerns in relation to the release of genetically-modified organisms into the

environment have replaced technological constraints as the major hurdles to be overcome with

genetically-modified bacteria, and bio-containment has emerged as an environmental priority

(75). Nevertheless, it would appear highly likely that future probiotic studies will be focussed

not only on exploiting their existing beneficial properties, but also their capacity to deliver a

broad range of specific factors, ranging from vitamins and antibodies through to strategically-

developed, genetically-modified agonists and inhibitors.

Probiotic mixtures and formulations

Simple ingestion of a broad spectrum of probiotics would appear to be a pragmatic approach

to cover a range of different indications and individual variations in host flora, although this

would assume that individual probiotic constituents are not mutually antagonistic.

Furthermore, it is a fundamental principle of therapeutic development that the properties and

optimal usage of individual components of any mixture or formulation should be

comprehensively determined before they can be recommended in combination. Undoubtedly,

however, the most compelling evidence for probiotic efficacy in IBD has been reported with a

combination of eight bacterial strains in the maintenance of remission of active ulcerative

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colitis (76) and prevention of pouchitis (77). This combination, termed VSL#3, comprises

four strains of lactobacilli and three strains of bifidobacteria, together with Streptococcus

thermophilus. Indeed, there is now a sizeable scientific literature on VSL#3 and its

application for a growing number of clinical disorders, with a recent report indicating

hydrolytic activity against gliadin polypeptides suggesting a possible application for VSL#3

in the control of coeliac disease (78).

Rachmilewitz et al (48) have reported anti-inflammatory effects of VSL#3 in murine

experimental colitis, mediated by Toll-like receptor (TLR9) signalling. Importantly, these

authors described that intragastric and subcutaneous administration of probiotic DNA

ameliorated the severity of experimental colitis, whereas methylated probiotic DNA, calf

thymus DNA, and DNase-treated probiotics had no effect. Moreover, colitis severity was

attenuated to the same extent by intragastric delivery of non-viable, gamma-irradiated, or

viable probiotics, suggesting that the protective effects of probiotics are mediated by their

own DNA rather than by their metabolites or ability to colonize the colon. The finding that

live micro-organisms were not required to attenuate experimental colitis holds significant

implications for the further development of probiotics as prophylactics or therapeutics in IBD.

The development of further probiotic combinations for IBD treatment appears highly likely,

although the current strategy will remain somewhat empirical until better-defined predictive

in vivo and in vitro systems have been developed.

The emergence of more compelling pre-clinical data from well-controlled animal model

studies is therefore proving to be a positive strategy for probiotic efficacy studies in

prospective IBD research. Such a strategy, in the absence of a rigorous, rapid throughput in

vitro screening assay, will become vital given the likely complexity of probiotic formulations

comprising mixtures of currently-characterised and newly-developed ‘designer’ probiotics.

To test the matrix of probiotic formulations in human clinical trials, even if conducted

strategically, would clearly be impossible.

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Prebiotics and Synbiotics

Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by

selectively stimulating the growth of bacterial species already established in the colon and

thus improve host health (79). These are usually of a poly- or oligo-saccharide nature with

studies largely confined to fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS) and

maltodextrin (80). The health interest of the Bifidobacterium genus is reflected in the

commonly-accepted definition of prebiotics: food ingredients that selectively stimulate the

growth and activity of bacteria in the gut, usually bifidobacteria (bifidogenic effect) and

lactobacilli, thereby producing health benefits.

Although an exhaustive review of prebiotics is beyond the scope of this review, the

importance of identified prebiotics in combination with dead or live probiotics (synbiotics), or

biologically active bacterial metabolites, should not be under-stated. For example, Kanauchi

et al (81) have recently described a synbiotic combination of germinated barley foodstuff

(GBF) and Eubacterium limosum (E. limosum) and its potential as an adjunctive treatment for

IBD. Although probiotic approaches for IBD include VSL#3, Nissle1917, Clostridium

butyricum, and Bifidobacterium-fermented milk, Eubacteria have not been studied to any

great extent. E. limosum is a commensal micro-organism that is promoted by GBF

administration, and its metabolites include butyrate, which can accelerate intestinal epithelial

growth and inhibit IL-6 production. GBF is therefore a prebiotic, and its unique

characteristics make it highly suitable for applications in IBD. GBF prolongs remission in

ulcerative colitis patients and also attenuates clinical activity in non-remissive colitic patients.

The further complexity of incorporating the increasing development and number of prebiotics

into the expanding range of newly-developed probiotic formulations described previously,

greatly increases the number of synbiotic combinations which await testing in pre-clinical

systems. However, although this enormous number of synbiotics and designer synbiotics may

be intimidating from a pre-clinical efficacy perspective, there exists the increased potential to

identify greater numbers of promising, efficacious candidate treatment formulations for IBD.

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Combining this strategy with genetic and genomic approaches to define IBD susceptibility

and probiotic responsiveness could ultimately result in individually-tailored probiotic-based

treatment approaches in IBD.

Personalised probiotics

Optimal selection of a probiotic may even need to take into account individual variations in

host diet and composition of gut flora. In this respect, the apparent influence of human genetic

variability on intestinal bacterial composition is particularly intriguing (82). Furthermore, it

seems unlikely that a single probiotic will be equally suited to all conditions. As a

consequence, selection of strains for disease-specific indications will be required (27). It is

beginning to become apparent that our definition of probiotics may be somewhat simplistic

since there are indications a probiotic under certain circumstances, may not be a probiotic at

all under altered physiological or pathogenic states, or even in different hosts. Different

probiotics have distinct properties, and not all models of experimental colitis respond to the

same probiotics (83). Almost certainly, there will not be a ‘one size fits all’ approach to

probiotic use in IBD. Personalised, designer probiotics and synbiotics could therefore be a

real therapeutic option for IBD sufferers in the future.

Conclusions

Probiotics continue to hold great promise for IBD treatment and prevention, but despite some

significant advances, it would be fair to say that this field of research is still in its infancy.

Understanding the mechanisms responsible for the beneficial effect of probiotics in

inflammatory bowel disease and experimental colitis will aid our understanding of the role of

endogenous and exogenous bacteria in IBD aetiology and pathogenesis. The finding that live

probiotics may not be essential for therapeutic effects, and that these effects may also be

obtained by the systemic route of administration, could have a major impact on the use and

manufacture of probiotics. Phenotypic and genomic characterisation of probiotic strains will

be required, together with clarification of their mechanisms of action across a broad range of

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rigorously-controlled clinical settings. Moreover, results of probiotic studies demonstrating

efficacy in a given clinical setting should not be extrapolated to other disease indications

without separate controlled assessments.

Over the past decade, there have been quantum advances in probiotic-based research in IBD.

Initial, largely anecdotal, reports of lactobacillus efficacy have been replaced by the

development of specific ‘designer’ probiotics, which together with identified prebiotics, could

result in a significant repertoire of designer synbiotics for IBD treatment. Such an array of

treatment options, combined with genetic advances in IBD susceptibility and probiotic

responsiveness, could eventually result in the exciting emergence of individually-tailored

probiotic-sourced formulations. The major challenge in the short-term will be the

development of rigorous in vivo and in vitro testing systems to rapidly determine the

suitability of any given probiotic-based formulation, not only to a specific condition, but also

to the individual.

Acknowledgements

The author would like to thank Associate Professor Ross Butler for his inspirational

contributions into the preparation of this review, Mr Mark Geier for his valued comments and

critique, and Miss Kerry Jolly for editorial assistance.

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Probiotics and Inflammatory Bowel DiseaseShort Title: Probiotics and Inflammatory Bowel Disease. Probiotics and IBD The intestinal microbiota and IBD aetiologyMechanisms of probiotic action in IBDAlthough perhaps a little simplistic, the ability for probiotics to prevent or combat infections may be partially- or entirely dependent upon mutual competitive metabolic interactions with potential pathogens, production of anti-microbial peptides such as bacteriocins (25) or inhibition of epithelial adherence and translocation by pathogens (26,27). Interestingly, Collado et al (28) reported that in general, bifidobacterial strains of animal origin demonstrated an improved capacity to inhibit and displace pathogens from human mucus than were human strains, further reporting that bifidobacteria were capable of producing antimicrobial peptides directed against Helicobacter pylori (29). Cross-species utilization of probiotics may therefore provide another important level of complexity for probiotic utility in clinical disorders such as IBD. In certain allergic disorders, including atopic eczema, probiotic influences on mucosal barrier function may be operative (30), whilst multiple mechanisms may account for anti-neoplastic effects (31). Probiotics, tolerance and the immune responseSince control of bowel inflammation has generally been recognised as paramount in IBD, it is perhaps not surprising that down-regulation of mucosal inflammation has been identified as the primary focus of both conventional and probiotic-targeted therapy, fuelled by the cumulative clinical experience with anti-TNF based treatment successes (32). Rodents and humans are normally tolerant to autologous microbiota, and an association between breakdown of this tolerance and the development of chronic intestinal inflammation has been demonstrated (32). Potentially, pathological responses to components of the intestinal flora may occur under normal physiological conditions, but these may be suppressed by immunoregulatory mechanisms. In a recent review by Thompson-Chagoyán et al (33), 11 models of IBD have been described, in which inflammation was found to be dependent on the presence of normal flora; the absence of normal flora being associated with non-appearance of the condition (34,35,36,37). This phenomenon has been reported across species (mice, rats and guinea pigs), and to occur in manipulated organisms such as transgenic mice with targeted deletion of the T-cell receptor (TCRα), that spontaneously develop colitis in response to the gut microbiota (38). Mucosal inflammation in rats and mice with induced IBD has also been reported to respond to treatment with broad-spectrum antibiotics (39). Moreover; in some animal model systems, colonisation with normal flora results in the rapid development of T-cell-mediated gut inflammation which can be transferred to other animals using activated T cells directed against enteric bacteria (40). Nevertheless, it appears that not all commensal bacteria have an equivalent ability to induce mucosal inflammation, which is also influenced by the host genetic background.Following cell surface recognition via Toll-like receptors (41), the anti-inflammatory effects of probiotics require signalling with the epithelium and hence, the mucosal immune system (42). Although transduction of bacterial signals into host immune responses presumably involves more than one pathway, NF-kappaB has been established as a central regulator of epithelial responses to invasive pathogens (43,44). Non-pathogenic components of the flora may attenuate pro-inflammatory responses by delaying degradation of IkappaB, the counter-regulatory factor to NF-kappaB (44). Indeed, it may be that probiotic bifidobacteria and lactobacilli may not use the same mechanism to achieve their anti-inflammatory effects as other signal transduction pathways begin to emerge. Clearly, a better understanding of the interplay between prokaryotes and eukaryotes, and subsequent effects on metabolic activity, will facilitate our knowledge of probiotic mechanisms. Bacterial adjuvants, including peptidoglycan, lipopolysaccharide, and DNA (CpG) bind to membrane-bound Toll-like receptors (TLR-2, 4, and 9. respectively), or cytoplasmic (NOD1 and NOD2) receptors (pattern recognition receptors), that activate nuclear factor-kappaB and transcription of many proinflammatory cytokines (45). Prokaryotic DNA perhaps represents the first description of a growing number of bacteria-sourced factors with the potential to alter host epithelial and mucosal immune responses. Un-methylated cytosine–guanine (CpG)-containing DNA, the ligand for Toll-like receptor 9 (TLR9), is a recently recognized microbial product with immuno-stimulatory and immuno-regulatory effects (46). TLR9 is expressed by many cell types located in the intestine, including epithelial cells and dendritic cells, and subcutaneous administration of immuno-stimulatory DNA has been reported to reduce the severity of experimental and spontaneous colitis in murine models of IBD (47) via a mechanism attributed to TLR9 signalling (48).A decrease in the secretion of pro-inflammatory cytokines, IFN-gamma, TNF-alpha and IL-12, and interference with bacterial adherence to the epithelium has been demonstrated following probiotic administration, associated with NF-kappaB inhibition, heat-shock protein induction and proteasome inhibition, although NF-kappaB induction has also been demonstrated (49,50). Unexpectedly, many of these beneficial effects have been achieved not only by live bacteria, but also by gamma-irradiated non-viable bacteria, bacterial DNA components and probiotic-cultured media (49,51). Investigations into probiotic supernatants and their therapeutic potential in IBD are therefore forming the basis for new directions in IBD research. In summary, although mechanisms of probiotic action may vary, depending on the experimental or clinical context, and depending on differences in the host and in the bacterial strain, the engagement with host immunity is pivotal to probiotic action in IBD. Probiotics as therapeutic agents in IBDThe human colon is a densely populated microbial ecosystem with several hundred bacterial species usually present with a total weight estimated to be several hundred grams (52). There are up to 1013–1014 total bacteria in the human intestinal tract, representing 10- to 20-fold more than the total number of tissue cells in the entire body, with most bacteria being obligate anaerobes, including clostridia, eubacteria, bacteroides groups and the genus bifidobacterium, such as Bifidobacterium bifidum and Bifidobacterium infantis (53).Probiotic efficacy has been confirmed in animal model studies by several investigators with different probiotic strains (37,53), although the use of probiotic therapy for IBD has only recently attracted serious interest from clinicians. Clinical trials in Crohn's disease with organisms, including lactobacillus (54) yeast (13), and coliforms (56), have been confounded by small patient numbers, differences in disease activity and variations in disease distribution (56). These deficiencies have been exacerbated by a growing, but poorly regulated, commercial market for probiotics, often linked with somewhat tenuous, or exaggerated, claims for health benefits. Historically, the stability, optimal dose-range, frequency of administration and vehicle for delivery, have rarely been determined. However, at present in Europe, well-designed and appropriately controlled trials of individual probiotic preparations are underway in both Crohn's disease and ulcerative colitis, to ascertain their efficacy in both active disease and in prevention of relapse.Individual probiotics and IBD treatmentThe predominant, potentially health-enhancing, bacteria in IBD are the bifidobacteria and lactobacilli, both of which belong to the lactic acid bacteria (LAB) group (57). These two genera do not include any significant pathogenic species and their dominance in the faeces of breast-fed babies is thought to impart protection against infection (58,59). Most commercially available probiotics meet minimum selection criteria including acid and bile resistance and survival during gastrointestinal transit, but an ideal individual probiotic strain for any given indication has still yet to be defined. Lactobacilli and bifidobacteria have traditionally been the most common candidates for probiotic-based treatments in IBD. Although there is a growing body of scientific literature and a wealth of anecdotal information supporting the utility of lactobacillus strains as therapeutic agents in a range of alimentary disorders, progress has perhaps been hampered by poorly-controlled associations between probiotic administration and clearly-defined clinical end-points. Recently, Hawrelak et al (60) conducted a systematic review of six clinical trials investigating the capacity for Lactobacillus rhamnosus GG to prevent the onset of antibiotic-associated diarrhoea. As data sources, these investigators employed computer-based searches of MEDLINE, CINAHL, AMED, the Cochrane Controlled Trials Register and the Cochrane Database of Systematic Reviews and the bibliographies of relevant papers and previous meta-analyses. Four of the six trials found a significant reduction in the risk of antibiotic-associated diarrhoea with co-administration of Lactobacillus GG; one trial reported a reduced number of days with antibiotic-induced diarrhoea, whilst the final trial found no benefit of Lactobacillus GG supplementation. These results are not unique in the context of retrospective probiotic studies, highlighting the need for additional research and the development of strictly-controlled predictive systems to clarify the effectiveness of Lactobacillus-based treatments beyond the effects of LGG in prevention of antibiotic-associated diarrhoea.Bifidobacterium, a member of the dominant microbiota (i.e. >108–109 colony forming unit (CFU)/g, represent up to 25% of the cultivable faecal bacteria in adults and 80% in infants. As probiotic agents, bifidobacteria have been studied for their efficacy in the prevention and treatment of a broad spectrum of animal and/or human gastrointestinal disorders, such as colonic transit disorders, intestinal infections, and colonic adenomas and cancer (10). Indeed, certain strains (eg Bifidobacterium animalis strain DN-173 010) have been reported to prevent or alleviate infectious diarrhoea through effects on the immune system and resistance to colonization by pathogens and there is some evidence that certain bifidobacteria may actually protect the host from pro-carcinogenic activity of intestinal flora (10).It should be noted, however, that probiotic properties have not been attributed solely to bacterial sources, since non-bacterial organisms, such as Saccharomyces boulardii, or even nematode parasites have been utilised for probiotic purposes (13,65) In summary, it seems unlikely that a single probiotic will be equally suited to all indications; selection of strains for disease-specific indications will be required, and it is in this capacity that well-conducted animal model studies will prove a valuable tool ConclusionsProbiotics continue to hold great promise for IBD treatment and prevention, but despite some significant advances, it would be fair to say that this field of research is still in its infancy. Understanding the mechanisms responsible for the beneficial effect of probiotics in inflammatory bowel disease and experimental colitis will aid our understanding of the role of endogenous and exogenous bacteria in IBD aetiology and pathogenesis. The finding that live probiotics may not be essential for therapeutic effects, and that these effects may also be obtained by the systemic route of administration, could have a major impact on the use and manufacture of probiotics. Phenotypic and genomic characterisation of probiotic strains will be required, together with clarification of their mechanisms of action across a broad range of rigorously-controlled clinical settings. Moreover, results of probiotic studies demonstrating efficacy in a given clinical setting should not be extrapolated to other disease indications without separate controlled assessments. Over the past decade, there have been quantum advances in probiotic-based research in IBD. Initial, largely anecdotal, reports of lactobacillus efficacy have been replaced by the development of specific ‘designer’ probiotics, which together with identified prebiotics, could result in a significant repertoire of designer synbiotics for IBD treatment. Such an array of treatment options, combined with genetic advances in IBD susceptibility and probiotic responsiveness, could eventually result in the exciting emergence of individually-tailored probiotic-sourced formulations. The major challenge in the short-term will be the development of rigorous in vivo and in vitro testing systems to rapidly determine the suitability of any given probiotic-based formulation, not only to a specific condition, but also to the individual.